Chemokines are a large family of proteins having a single polypeptide chain of about 70–100 amino acids that act through G-protein coupled receptors to regulate a variety of biological processes such as the recruitment of immune cells to the site of inflammation, angiogenesis, hematopoiesis, and organogenesis. The name “chemokine” is derived from the words “chemoattractant” and “cytokine” and stems from the observation that most members of the family have leukocyte chemoattractant and cytokine-like activity. The primary function of chemokines is thought to be as regulators of the processes of leukocyte trafficking during immunity and inflammation. More than 50 members of the chemokine family are known (Wolpe & Cerami, 1989, FASEB J. 3:2565–2573; Baggiolini et al., 1994, Adv. Immunol. 55:97–179; Locati & Murphy, 1999, Ann. Rev. Med. 50:425–440; Luster, 1998, New Eng. J. Med. 338:436–445).
Chemokines have been divided into subfamilies based upon the number and spacing of conserved cysteine residues. The CC and CXC subfamilies have four cysteines, of which the first two are either adjacent (the CC subfamily) or separated by a single amino acid (the CXC subfamily). Both of these subfamilies have multiple members, which carry out their biological roles through interaction with a wide variety of chemokine receptors. In addition, there are two additional chemokine families: the C family, with no intervening amino acids, and the CXXXC family with three intervening amino acids.
There are at least 15 types of chemokine receptors, with each type of receptor generally being capable of binding to a particular subfamily of chemokines. The names of the receptors reflect this subfamily-restricted binding specificity. Thus, the receptors that bind CXC chemokines are known as the CXCRs; those receptors that bind CC chemokines are known as CCRs. The general subfamily-specific name is followed by a number, reflecting the order of discovery of a particular receptor. Thus, CXCR4 was the fourth receptor found that was specific for the CXC subfamily of chemokines. Chemokine receptors belong to the large class of rhodopsin-like, 7-transmembrane (7TM), G-protein coupled receptors (GPCRs). They are generally coupled to Gi-type proteins (Murphy, 1994, Ann. Rev. Immunol. 12:593–633; Murphy, 1996, Cytokine Growth Factor Rev. 7:47–64; Yoshie et al., 1997, J. Leukocyte Biol. 62:634–644).
An important development in chemokine research occurred with the discovery that certain chemokines could suppress HIV-1 virus infection in vitro. This effect was determined to be due to blockage of the interaction of the virus's envelope glycoprotein (Env) with certain chemokine receptors. The primary receptor for all strains of HIV-1 is CD4, a membrane protein found on T cells and certain other cells, but strain-specific chemokine receptors are also required as coreceptors. The interaction of Env, CD4, and the chemokine receptor results in the fusion of the viral and target cell membranes (Feng et al., 1996, Science 272:872–877; Cocchi et al., 1995, Science 270:1811–1815; Berger, 1997, AIDS 11(Suppl. A):S3–16; Rucker et al., 1997, J. Virol. 71:8999–9007; Wyatt & Sodroski, 1998, Science 280:1884–1888).
The most widely used chemokine coreceptors for HIV-1 are CCR5 and CXCR4. Virtually all primary HIV-1 isolates use either CXCR4, CCR5, or both (Zhang et al, 1996, Nature 383:768; Connor et al., 1997, J. Exp. Med. 185:621–628; Bjorndal et al., 1997, J. Virol. 71:7478–7487; Scarlatti et al., 1997, Nature Med. 3:1259–1265; Bazan et al., 1998, J. Virol. 72:4485–4491). There is a curious and as yet incompletely explained tropism to HIV-1 isolates. CCR5-specific strains are associated with early infection and the period of clinical latency while CXCR4 -specific strains are associated with later stages of infection that include immune system collapse and AIDS. The earlier strains are able to infect primary macrophages in vitro while the later strains are able to infect T cell lines. This cellular tropism is understood to be due to the different patterns of chemokine receptor expression in the two target cell types; macrophages express CCR5 while T cells express CXCR4. In general, HIV-1 tropism can be explained by the ability of an HIV-1 isolate's Env to recognize a particular chemokine receptor and by the expression patterns of these chemokine receptors on CD4+ target cells. In fact, tropism has been re-defined based upon the usage of CCR5 (R5 tropic virus), CXCR4 (X4 tropic virus), or both CCR5 and CXCR4 (R5x4). Although helpful, this simplified picture is subject to occasional complications. For example, macrophages express CXCR4 in addition to CCR5. The block to X4 tropic virus in macrophages occurs post entry. Although CCR5 and CXCR4 are the principal coreceptors utilized in vivo, many other chemokine receptors and related orphan receptors also have HIV-1 coreceptor activity in vitro, including CX3CR1, CCR8, CCR2, CCR3, STRL33/BONZO, GPR1, GPR15, and APJ (Berger, 1997, AIDS 11 (Suppl. A):S3–16; Rucker et al., 1997, J. Virol. 71:8999–9007).
The role of CCR5 in the pathogenesis of HIV-1 infection and the development of AIDS has been clearly demonstrated. A mutant allele with a 32 base pair deletion that results in a truncated, inactive receptor (CCR5 d32) is found in homozygous form about 20-fold less frequently in HIV-1 infected people than in the general population, indicating a substantial protective effect (Liu et al., 1996, Cell 86:367–377; Dean et al., 1996, Science 273:1856–1862). Other polymorphisms in chemokines or their receptors have been found to be associated with various levels of resistance to HIV-1 pathogenesis. Of special note is a polymorphism in the 3′ untranslated region of SDF-1 mRNA. SDF-1 is a chemokine ligand of CXCR4 (Winkler et al., 1998, Science 279:389–393). In addition to molecular epidemiology studies, much in vitro evidence strongly suggests that chemokine receptors play a role in HIV-1 pathogenesis. For example, the CCR5 ligands MIP-1α, MIP-1β, and RANTES are able to suppress HIV-1 replication in certain cultured cells (Cocchi et al., 1995, Science 270:1811–1815). Furthermore, a low molecular weight bicyclam compound, AMD3100, inhibits HIV-1 replication in SCID-hu mice (Datema et al., 1996, Antimicrob. Agents Chemother. 40:750–754). AMD3100 acts by blocking virus interaction with the CXCR4 coreceptor (Schols et al., 1997, J. Exp. Med. 186:1383–1388).
Since chemokine receptors are involved in HUV-1 pathogenesis, it is of great interest to identify substances that can inhibit the interaction of HIV-1 Env proteins and chemokine coreceptors. However, to date, satisfactory methods of identifying such inhibitors are lacking.
Nussbaum et al., 1994, J. Virol. 68:5411–5422 and Feng et al., 1996, Science 272:872–877 disclosed a system in which a first cell was infected with a vaccinia virus encoding an HIV envelope protein as well as bacteriophage T7 RNA polymerase. A second cell expressed CD4, a chemokine receptor, and the E. coli LacZ gene under the control of a T7 promoter. Upon fusion of the two cells, the T7 RNA polymerase from the first cell transcribed the LacZ reporter gene from the second cell and the activity of the product of the reporter gene was measured. Inhibitors of HIV fusion were identified by their ability to suppress readout from the reporter. This system has many disadvantages: generally, hours must pass before readout from the reporter occurs; the use of vaccinia virus requires Class 2 biosafety conditions; agents which block viral assembly and replication, such as araC or rifamycin, must be used; and, because the assay depends on the transcriptional activity of the T7 promoter, it will identify inhibitors of transcription in general, irrespective of whether such inhibitors affect fusion.
Several assays based upon fluorescence have been developed. Weiss et al., 1996, AIDS 10:241–246 labeled lymphocytes (cells that grow in suspension) with intracellular fluorescent dyes and mixed the labeled lymphocytes with unlabeled adherent cells under conditions where fusion could occur. The occurrence of fusion was monitored by scanning microscopic fields for the presence of fluorescent adherent cells. This assay did not make use of fluorescence resonance energy transfer and suffered from the drawback that the two cell types used must be morphologically distinct. Also, the assay is difficult to quantitate.
A similar assay was developed in which the two cells to be fused were labeled with two different fluorescent dyes, with the dyes having overlapping emission and excitation spectra (Litwin et al., 1996, J. Virol. 70:6437–6441 (Litwin); International Patent Publication WO 96/41020). The first and second dyes are chosen so that the emission spectrum of the first overlaps the absorption spectrum of the second. In the absence of fusion between the two cells, little fluorescence resonance energy transfer (FRET) will be observed since the two dyes are not likely to be physically close to one another. When the two cells are mixed under conditions such that they fuse, however, the two dyes are brought into close proximity within the fused membranes, thus allowing FRET between the two dyes to occur. If the two cells are mixed in the presence of an inhibitor of fusion, little or no FRET will be observed. Screening a collection of compounds for those compounds capable of diminishing or preventing FRET will identify compounds that are inhibitors of fusion. The assays described in Litwin and International Patent Publication WO 96/41020 require the use of two physically separate dyes since a different dye must be incorporated in each cell's membrane. Also, Litwin and International Patent Publication WO 96/41020 do not disclose the importance of chemokine receptors in the fusion process. Thus, the success of the methods described in Litwin and International Patent Publication WO 96/41020 depends on the chance selection of a cell type that coexpresses CD4 and an appropriate chemokine receptor. In addition, this assay requires the separate labeling of two different cell types, each with a different fluorescent dye.
An extremely sensitive assay for studying signal transduction that is based on FRET is disclosed in Zlokamik et al., 1998, Science 279:84–88 (Zlokarnik) and also in U.S. Pat. No. 5,741,657. The assay disclosed in Zlokarnik and U.S. Pat. No. 5,741,657 is designed for the study of transcriptional activation that arises as a result of intracellular signaling pathways that are activated by ligand-receptor interactions. The assay employs a plasmid encoding β-lactamase under the control of an inducible promoter. This plasmid is transfected into cells together with a plasmid encoding a receptor for which it is desired to identify agonists. The inducible promoter on the β-lactamase is chosen so that it responds to at least one intracellular signal that is generated when an agonist binds to the receptor. Thus, following such binding of agonist to receptor, the level of β-lactamase in the transfected cells increases. This increase in β-lactamase is made measurable by treating the cells with a cell-permeable dye that is a substrate for β-lactamase. The dye contains two fluorescent moieties. In the intact dye, the two fluorescent moieties are close enough to one another that FRET can take place between them. Following cleavage of the dye into two parts by β-lactamase, the two fluorescent moieties are located on different parts, and thus can diffuse apart. This increases the distance between the flourescent moieties, decreasing the amount of FRET that can occur between them. It is this decrease in FRET that is measured in the assay. Zlokarnik used this assay to identify inhibitors of the M1 muscarinic receptor. Zlokamik did not disclose the use of this assay to study membrane fusion in general and the interaction of HIV-1 and target cells in particular.